Выделение и культуры клеток нервного гребня из эмбриональных мышей нервной трубки

Summary

Выделение эмбриональных нервного гребня из нервной трубки облегчает использование<em> В пробирке</em> Методы изучения миграции, самообновления и multipotency из нервного гребня.

Abstract

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types ^1-3. NC also has the unique ability to influence the differentiation and maturation of target organs^4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone.

NC multipotency was first described from explants of the avian neural tube^7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo^10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro.

To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors^11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties^13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors^11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter²³.

The method presented here is adapted from protocols optimized for the culture of rat NC^11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC^2,24-28.

Protocol

1. Подготовка пластин Использование стерильных во все времена. Подготовить фибронектин (FN) путем разбавления 100 мкл плазмы человека запас FN в конечном объеме 3,3 мл в PBS Дульбекко (DPBS). Конечная концентрация 30 мкг / мл, и это можно хранить при температуре 4 ° С в течение 1 недели. …

Discussion

Особое внимание следует обратить на стадии развития эмбриона, чтобы обеспечить успех этого подхода. Подсчет сомитов ранних эмбрионов мыши имеет решающее значение как для стадии эмбриона в соответствие мусора и правильно определить регионы нервной трубки для изоляции. Изменение одно?…

Materials

Reagent	Company	Catalogue number	Comments
DMEM (low glucose)	Gibco/Invitrogen	11885
Neurobasal Medium	Gibco	21103
BSA	Sigma	A3912-10G
dPBS	Gibco	14190-144
IGF1	BD Biosciences	354037	Store in 50 μg/mL aliquots at -20°C.
bFGF	BD Biosciences	354060	Store in 25 μg/mL aliquots at -20°C.
Fibronectin	Gibco	33016-015	Stored in 1mg/mL aliquots at -20°C.
Retinoic acid	Sigma	R2625	Store in 35 μg/ml aliquots after reconstituting in ethanol at -20°C.
2-mercaptoethanol	Sigma	D-5637
N2 supplement	Gibco	17502-048
B27 supplement	Gibco	17504-044
Steriflip 0.22 μm filters	Millipore	SCGP00525
Penicillin-streptomycin	Invitrogen	15140122
0.20 μm filters	Corning	431219
Syringes (for filtration)	BD Biosciences	301604
Four well plates	Thermo Fisher Scientific	176740
Collagenase/Dispase	Roche	269 638	Activity varies by batch. Store in 100 mg/mL aliquots at -20°C.
Insulin needles (29½ gage)	Becton Dickson	309306
Hypoxia Chamber	Billups-Rothenberg
Oxygen Analyzer	Billups-Rothenberg
Forceps #5	Fine Science Tools		For removing uterus and decidua.
Trypsin-EDTA (0.25%)	Gibco	25200